Liver and kidney play important roles in the metabolism and excretion of biologically foreign compounds and drugs out of bodies. Tubule cells and hepatocytes belong to epithelial cells with polarities. It is supposed that some of anionic substances are taken up through the basolateral membranes into kidney and liver by transporters, while the organic anions generated metabolically in cells are excreted by transporters.
The uptake of organic anions through the basolateral membranes of tubule cells and hepatocytes have been investigated so far in experiment systems using isolated organ perfusion protocols, dissected cells and membrane vesicles. According to such conventional methods, however, the detailed analysis of the transport of organic anions through the basolateral membranes has been difficult. Accordingly, it has been desired to isolate the transporters per se and analyze the properties of transporters in detail.
Alternatively, plural experimental results suggestively indicate the presence of the transport of organic anions in brain. The transport of organic anions in brain is supposed to function for the extracerebral excretion of endogenous and exogenous organic anions.
Although the transport of organic anions in brain is speculated to play an important role in the elimination of endogenous anions and foreign compounds from brain, the detail of the transport therein is more ambiguous than the transport in kidney and liver, due to the difficulty in physiological experiments therein.
Based on these backgrounds, the screening of the organic anion transporter molecules per se has been actively carried out in 1990 and thereafter. Consequently, two organic anion transporters derived from the basolateral membrane of liver have been isolated until the last year. (Hagenbuch, B. et al., Proc. Natl. Acad. Sci. USA, Vol. 88, pp. 10629-33, 1991; Jacquemin, E. et al., Proc. Natl. Acad. Sci. USA, Vol. 91, pp. 133-7, 1994)
The present inventors independently isolated an organic anion transporter OAT1 responsible for the most important function in the organic anion transport in kidney successfully last year (Sekine, T., et al., J. Biol. Chem., Vol. 272, pp. 18526-9, 1997) and already filed the patent application thereof. OAT1 is a transporter capable of transporting a great number of organic anions with different chemical structures and is also involved in the transport of various anionic drugs. OAT1 is expressed in a specific manner to kidney, while OAT1 is very slightly expressed in brain except kidney.
Recently, the inventors have further identified a liver-specific organic anion transporter (OAT2) with about 40% homology to OAT1 in terms of amino acid level (FEBS letter, Vol. 429, pp. 179-182, 1998) (Japanese Patent Application No. 169174/1998).
The isolation and identification of OAT1 and OAT2 indicates that these organic anion transporters form a family. Additionally because OAT2 is expressed specifically in liver, it is suggested that the family is not kidney-specific but is expressed in various organs.
As described insofar, it is suggested that an organic anion transport system is present in brain, but the OAT1 expression in brain is quite slight while OAT2 is not present therein. Based on these findings, the inventors have anticipated the presence of an unknown transporter responsible for the organic anion transport in brain.
Alternatively, the organic anion transport in the basolateral membrane of liver is complicated; particularly, the efflux flow of conjugated substances (many of the conjugated substances are organic anions) generated at a vast scale in hepatocytes into blood has not yet been known. The organic anion transport in liver cannot sufficiently be described on the single basis of the organic anion transporters including OAT2. Hence, the presence of an unknown transporter is suggested.
The inventors isolated the organic anion transporter OAT1 serving as the most important role in the organic anion transport in kidney (Sekine, T. et al., J. Biol. Chem. Vol. 272, pp. 18526-9, 1997). Based on the structural similarity to OAT1, the inventors identified a liver-specific organic anion transporter (OAT2) (Sekine, T., at al., FEBS letter, Vol. 429, pp. 179-182, 1998). The inventors already reported additionally (Sekine, T., at al., J. Biol. Chem., Vol. 272, pp. 18526-9, 1997) that OAT1 had low homology to an organic cation transporter OCT1 (Grundemann, D. et al., Nature, Vol. 372, pp. 549-52, 1994).
Taking account of these evidence, the inventors identified a sequence common to OAT1, OAT2 and OCT1 and prepared a degenerate primer based on the sequence. By using the degenerate primer, the inventors identified a novel cDNA fragment with low homology to OAT1, OAT2 and OCT1 from rat brain mRNA by RT (reverse transcript)-PCR (polymerase chain reaction) method. By using the cDNA fragment, a cDNA never reported yet was discriminated from the rat cDNA library. The resulting protein was designated cerebral type organic anion transporter OAT3 as a third member of the OAT family.